Diterpenoids target SARS-CoV-2 RdRp from the roots of Euphorbia fischeriana Steud

Introduction Currently, the development of new antiviral drugs against COVID-19 remains of significant importance. In traditional Chinese medicine, the herb Euphorbia fischeriana Steud is often used for antiviral treatment, yet its therapeutic effect against the COVID-19 has been scarcely studied. Therefore, this study focuses on the roots of E. fischeriana Steud, exploring its chemical composition, antiviral activity against COVID-19, and the underlying basis of its antiviral activity. Methods Isolation and purification of phytochemicals from E. fischeriana Steud. The elucidation of their configurations was achieved through a comprehensive suite of 1D and 2D NMR spectroscopic analyses as well as X-ray diffraction. Performed cytopathic effect assays of SARS-CoV-2 using Vero E6 cells. Used molecular docking to screen for small molecule ligands with binding to SARS-CoV-2 RdRp. Microscale thermophoresis (MST) was used to determine the dissociation constant Kd. Results Ultimately, nine new ent-atisane-type diterpenoid compounds were isolated from E. fischeriana Steud, named Eupfisenoids A-I (compounds 1-9). The compound of 1 was established as a C-19-degraded ent-atisane-type diterpenoid. During the evaluation of these compounds for their antiviral activity against COVID-19, compound 1 exhibited significant antiviral activity. Furthermore, with the aid of computer virtual screening and microscale thermophoresis (MST) technology, it was found that this compound could directly bind to the RNA-dependent RNA polymerase (RdRp, NSP12) of the COVID-19, a key enzyme in virus replication. This suggests that the compound inhibits virus replication by targeting RdRp. Discussion Through this research, not only has our understanding of the antiviral components and material basis of E. fischeriana Steud been enriched, but also the potential of atisane-type diterpenoid compounds as antiviral agents against COVID-19 has been discovered. The findings mentioned above will provide valuable insights for the development of drugs against COVID-19.


Introduction
In late 2019, viral infections spurred by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) profoundly impacted global human existence and productivity (Hu et al., 2021).As of early March 2024(World Health Organization, 2024), the World Health Organization (WHO) documented 775 million confirmed infections and 7.04 million deaths worldwide.Despite accessible medications and ongoing vaccination drives, viral mutations and waning immune responses have hampered vaccine efficacy, leading to re-infections (Sievers et al., 2022).SARS-CoV-2, an optimistic single-stranded RNA virus (Gorbalenya et al., 2020), employs RNA-dependent RNA polymerase (RdRp) for genome replication and gene transcription (Snijder et al., 2016).RdRp, as a pivotal enzyme in the viral life cycle, stands as a recognized target for antiviral medications (te Velthuis, 2014).Human life and productivity remain imperiled, and the development of targeted COVID-19 medications is urgently needed (Feikin et al., 2022).Natural products have long been esteemed as pivotal sources of medications for diverse ailments.Euphorbia is the largest genus of the Euphorbiaceae, one of the largest families of higher plants (Li et al., 2009).Euphorbia fischeriana Steud, a member of the genus Euphorbia (Euphorbiaceae), is a perennial herb, and the entire plant is poisonous and is grown primarily in Mongolia, Eastern Siberia, and China (Flora of China Editorial Committee, 1997).E. fischeriana Steud serves prominently in traditional Chinese medicine, the roots of which has a long history of traditional use for treating conditions such as edema, ascites, cough, and cancer, and also exhibits prowess in combating viral infections (Pharmacopoeia Commission of PRC, 2020).Its diterpenoids possess a spectrum of pharmacological activities, including antiviral, anti-tumor, antibacterial, anti-inflammatory, and other therapeutic properties (Li et al., 2021) (Figure 1).In traditional Chinese medicine, E. fischeriana Steud is an important antiviral herb.During the outbreak of the COVID-19 pandemic, efforts have been made in some regions of China to utilize traditional Chinese herbal remedies, including E. fischeriana Steud, to treat COVID-19 infections, with some degree of success (Dai et al., 2020).Through literature review, it was found that the material basis of E. fischeriana Steud's anti-COVID-19 activity, specifically the antiviral activity of its natural compounds and related mechanisms, has not been studied.Therefore, we selected E. fischeriana Steud as the research subject to investigate its chemical composition.We employed the latest techniques in pharmaceutical chemistry research, such as molecular docking and microscale thermophoresis (MST), to evaluate its potential anti-SARS-CoV-2 effects and related targets.Through in vitro cellular experiments, we identified ent-atisane-type diterpenoid compounds and potential targets among the anti-COVID-19 active herbal constituents, providing new insights for the development of COVID-19 therapeutic drugs.We have successfully isolated and identified nine diterpenoids with a novel structure from the roots of E. fischeriana Steud (Figure 2); in addition, we evaluated the anti-SARS-CoV-2 activity of the compounds and explored the targets and mechanisms of action.1, 2) spectra of 1 displayed 22 carbon signals and resonances attributable to four methyls, eight methylenes, five methines (one oxygenated methine), and five quaternary carbons (one carbonyl and two oxygenated carbons), corresponding to the units in its 1 H NMR data (Table 1).The 1 H-1 H COSY spectrum of this substance shows the existence of correlations of H-1/H-2 and H-4/H-5.Furthermore, analysis of the HMBC spectrum uncovered correlations of H-1/C-2, C-3 (d C 213.8), C-5 (d C 55.5), C-10, and H-4/C-10, H-5/C-3, showing the presence of an A ring with the carbonyl group at C-3.The 1 H-1 H COSY spectrum showed a correlation between CH 3 -18/H-4/H-5, and the HMBC spectrum showed a correlation between H-18/C-3, C-4, and C-5, H-1, H-5/C-20, indicating that C-18 (d C 11.7) and C-20 (d C 14.8) have a methyl group; C-20 is a horn methyl group.Compound 1 was one methyl less than ent-3-oxoatisan-16a, 17-acetonide (Yan et al., 2018), suggesting the absence of CH 3 -19 in 1.The 1 H-1 H COSY (Figure 3B) spectrum showed the correlation of H-9/H-11/H-12 and H-12/H-13/H-14, and the HMBC spectrum showed the correlation of H-11/C-16, C-10, which, combined with the HSQC (Tables 1, 2) (d C 67.4, C-11, d H 4.66, H-11), indicated an additional    Ka radiation, Flack parameter 0.01 (11)] (Figure 3A).Analysis of the structure shows that the absolute configurations at the stereogenic centers in compound 1 is 4R,5S,8S,9S,10R,11S,12S,16S.
The experimental findings revealed the presence of compound 1 in both the methanol and ethyl acetate extracts, with the ethyl acetate extraction layer containing a higher quantity of compounds than the methanol extract (Table 4).This substantiates that compound 1 is not an artificial product but rather a natural plant-derived compound.

Bioassay results for compound 1
We performed cytopathic effect assays of SARS-CoV-2 using Vero E6 cells with remdesivir as a positive control to further understand the biological activity of compound 1.In DMSO (Figure 5Ba) as naive control, the Vero E6 cells were morphologically intact.SARS-CoV-2-infected Vero E6 cells showed a morphological deformation with chromatin condensation and karyopycnosis (Figure 5Bb), demonstrating a significant cytopathic effect.The addition of compound 1 reduced the number of cells that developed lesions in comparison to before (Figure 5Bc), but not as much as remdesivir (Figure 5Bd).Thus, assays of cytopathic effect clearly show the anti-SARS-CoV-2 The UPLC-MS/MS results for sample.3 Materials and methods

General
One-and two-dimensional NMR spectra were determined by Bruker 500-MHz and 600-MHz NMR instruments with the internal standard: TMS.The chemical shifts d were expressed in parts per million (ppm), and the coupling constant J was expressed in Hz.The CD spectra were measured on a photophysical circular dichroism spectrometer (Applied Photophysics, Leatherhead, Surrey, UK).HR-ESI-MS data acquisition was performed in positive mode on an Agilent 1290 UPLC/6540 Q-TOF mass spectrometer.The UV spectra were detected on a Shimadzu UV-2401A UV spectrometer, the specific spin data were detected on a JASCO DIP-370 digital spinometer, and the IR spectra were detected on a Tenor 27 infrared spectrometer with KBr pressurization assay.Single-crystal x-ray diffraction experiments were detected on a Bruker APEX DUO diffractometer with a copper target.Semipreparative HPLC was performed on an Agilent 1260 apparatus equipped with a UV detector and a Zorbax SB-C-18 (Agilent, 9.4 mm × 25 cm) column.Column chromatography (CC) was performed using silica gel (200-300 mesh and H, Qingdao Marine Chemical Co. Ltd., Qingdao, China) and RP-C18 gel (40-63 mm, Merck, Darmstadt, Germany).Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co. Ltd., Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.All solvents were distilled prior to use.

Plant material
The roots of E. fischeriana Steud were collected in September 2015 from Xianggelila City, Yunnan Province, People's Republic of China.The plant samples were identified by Prof. Xun Gong of the State Key Laboratory of Phytochemistry and Plant Resource in West China.Voucher specimens (HXJ20150915) were deposited at Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS).

Compound naming rule
The compound name consists of a partial letter of the plant's Latin name (E. fischeriana Steud) and the suffixes of the terpenoids.

X-ray crystallographic data for compound 1
Colorless crystals of 1 were obtained by recrystallization in MeOH at room temperature.X-ray crystal data were acquired on a Bruker APEX-II CCD detector with graphite monochromated Cu Ka radiation (l = 1.541, 78 Å).The structure of 1 was directly elucidated using SHELXL-97 (Sheldrick 2008) and refined by the full-matrix least-squares difference Fourier method.The x-ray data of 1 have been deposited at the Cambridge Crystallographic Data Center.

Assays of cytopathic effect of compound 1
Vero E6 cells were seeded in 96-well plates and grown overnight.Cells were incubated with SARS-CoV-2 at 37°C for 2 h and infected at a multiplicity of infection of 0.1.Then, the cells were incubated with the maintenance medium in compound 1 (1 mg, 20 mM).Remdesivir (4 mM) was used as positive controls and DMSO solution was used as naive control.After 72 h, cell viability was then assessed using colorimetric MTS assays (Promega Corp.) as described by the manufacturer.Thereafter, the cells were photographed using a microscope.

Molecular docking of compound 1
Autodock tool 1.5.6 software was used to perform operations such as acceptor polarization of hydrogen, Gasteiger charge distribution, and removal of water molecules for 7BV1 and smallmolecule ligands.Set the docking central coordinates of the Autodock Vina software to center_x = 131.622,center_y = 135.777,center_z = 121.114.The docking box size is 126 Å, the exhaustiveness value of the search parameter is 10, the top nine conformations are output according to the docking score, and the default value is selected for the rest of the parameters.Finally, the docking results were visualized by PyMOL software.

Microscale thermophoresis of compound 1
Purified SARS-CoV-2 RdRp protein was subjected to NHS (lysine labeling method) labeling.One hundred microliters of 10 µM protein and lysine labeling reagent was incubated in a dark environment for 30 min.The initial concentration of compound 1 (1 mg) was set at 20 mM, and it was subsequently diluted to a concentration of 100 µM using PBS-T buffer, with 16 gradient dilutions of 100 µM.Twenty microliters of compound 1 solution was added to PCR tube 1 and 10 µL of PBS-T buffer was added to the remaining 15 PCR tubes.Ten microliters of solution was pipetted from tube 1 and added to tube 2 and mixed; this process was repeated for tubes 3 to 16, the serial dilution was completed sequentially, and 10 mL was discarded in the last tube.Ten microliters of SARS-CoV-2 RdRp protein solution was added to each PCR tube and mixed well, a capillary was used to aspirate the sample, and the MST experiment was performed.The instrument was set to a medium MST power, and the K d values and binding curves of compound 1 and the SARS-CoV-2 RdRp protein were ultimately obtained.The dilution and assay steps were repeated on two separate occasions.

Discussion
Plants, with their complex secondary metabolism, produce a wide range of compounds and offer significant advantages in the treatment of infectious diseases.Throughout history, phytotherapy has been utilized during epidemics such as the Black Death, smallpox, tuberculosis, malaria, and Spanish flu, providing valuable references for mankind on the safety and effectiveness of plant-based treatments (Garcia, 2020).Developing anti-SARS-CoV-2 drugs based on existing antiviral plants that have a proven track record could streamline the clinical trial process and expedite the identification of potential plant inhibitors (Pandey et al., 2020).In this study, we focused on extracting and isolating compounds from the roots of E. fischeriana Steud with potential activity against SARS-CoV-2.Nine undescribed ent-atisane type diterpenoids were successfully isolated from this plant.The elucidation of their configurations was achieved through a comprehensive suite of 1D and 2D NMR spectroscopic analyses as well as x-ray diffraction.Atisane-type diterpenoids belong to the tetracyclic diterpenoid family.They possess a bicyclo[2.2.2]octane ring system, decorated with methyl groups at C-4, C-10, and C-16.The most frequently oxidized positions of the ent-atisane skeleton are C-3, C-16, and C-17; C-16 and C-17 are typically in the form of an olefin (Drummond et al., 2021).The double bond at C-16 and C-17 is oxidized to a terthydroxyl group at C-16, which undergoes further oxidation to form an acetone dimethyl acetal to finally form the compound we obtained in this study.Many of the ent-atisane diterpenoids have antiviral activity, including anti-influenza A virus (Zhang et al., 2024), anti-HIV-1 (Yan et al., 2018), and anti-human rhinovirus 3 (Wang et al., 2018).Notably, in our study, one of these compounds exhibited promising anti-SARS-CoV-2 activity, and cytopathic effect assays confirmed the anti-SARS-CoV-2 activity of compound 1.
Molecular docking plays a crucial role in the search for antiviral compounds within various plant extracts (Pandey et al., 2020).Using this way of thinking, based on existing antiviral plants, scientists have already employed an integrated approach combining network pharmacology analysis, molecular docking, LC-MS analysis, and bioassays to uncover the potential ingredients of Scutellariae radix for SARS-CoV-2 (Liu et al., 2022).In our study, molecular docking predicted that compound 1 has an affinity for RdRp, with a binding energy of −8.0 kcal/mol.Based on the molecular docking results, our study further investigated the affinity between compound 1 and RdRp using MST, and these findings suggest that compound 1 could serve as a potential therapeutic target against SARS-CoV-2 RdRp.The search for potential antiviral agents targeting SARS-CoV-2 RdRp remains a subject of ongoing research.For instance, the polyphenolic compound gossypol could directly block SARS-CoV-2 RdRp, thereby inhibiting SARS-CoV-2 replication in cellular and mice models of infection (Wang et al., 2022).Other compounds like quercetin and procyanidins have also demonstrated promising inhibitory effects on SARS-CoV-2 RdRp through in vitro enzyme assays (Jin et al., 2022;Munafò et al., 2022).The diversity of plant-derived compounds is illustrated by the fact that different types of compounds can act on the same targets.Based on our work, compound 1 also appears promising as a SARS-CoV-2 RdRp inhibitor.In addition, structural modifications may further enhance the anti-SARS-CoV-2 activity of compound 1.
Medicinal plants have long been used to treat infectious diseases and have been vital to human society.As our understanding of plant science grows, we will continue to discover new plant-derived drugs.In order to fully utilize the role of medicinal plants, we need to apply modern technology and a great deal of multidisciplinary research.The current study, which is a tiny portion of plant science research, aims to offer some new scientific foundation.

Conclusions
In conclusion, this study conducted a preliminary exploration of the material basis, active compounds, and related targets of E. fischeriana Steud against SARS-CoV-2.Nine previously unreported ent-atisane-type diterpenoid compounds were isolated from the roots of E. fischeriana Steud, and their activities, targets, and mechanisms against SARS-CoV-2 were investigated.Cell pathology experiments confirmed that ent-atisane-type diterpenoid compound 1 exhibited certain anti-SARS-CoV-2 activity.Compound 1 was predicted to bind to RdRp through high-throughput virtual screening.Subsequently, using MST technology, the affinity between compound 1 and RdRp was tested, revealing that compound 1 could form a stable complex with RdRp protein, with a K d value of 31.13 mM.RdRp protein was preliminarily identified as the target of compound 1 against SARS-CoV-2.This work expands the research achievements of E. fischeriana Steud and its diterpenoid components in the field of antiviral research, providing valuable references for the discovery of potential anti-SARS-CoV-2 targets and mechanisms.Additionally, it enriches the library of antiviral active compounds against SARS-CoV-2 and clarifies the potential of ent-atisane-type diterpenoid compounds in antiviral research, offering new insights for COVID-19 drug development.

Compound 1 ,
white crystals, has the molecular formula C 22 H 34 O 4 as determined from a high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) peak at m/z 363.2529 [M+H] + (calcd.C 23 H 35 O 4 , 363.2530).The molecular formula indicates six indices of hydrogen deficiency.The 13 C NMR and DEPT (Tables

FIGURE 1
FIGURE 1Euphorbia fischeriana Steud plant pictures and other information.
FIGURE 5 (A) MST analysis results of compound 1.The linear fit is close to S, and the K d = 31.13mM.(B) Cytopathic effects of SARS-CoV-2-infected Vero E6 cells.(a) Vero E6 cells were treated with a DMSO solution.(b) Vero E6 cells were then infected with SARS-CoV-2; (c) SARS-CoV-2 + Remdesivir; (d) SARS-CoV-2 + compound 1 (because the compounds were screened in different batches, 2 in the figure actually represents compound 1; concentration was 20 mM).

FIGURE 6
FIGURE 6 Structure of compounds 1-9 bound to SARS-CoV-2 Rdrp.The compound is denoted in red.The left shows the state of the compound in the substrate-binding pocket of the SARS-CoV-2 Rdrp.The right shows the co-crystal structures of the compound with SARS-CoV-2 Rdrp.
a Chemical shifts (ppm) referenced to solvent peak (d C 77.16 in CDCl 3 ) at 126 MHz.b Chemical shifts (ppm) referenced to solvent peak (d C 49.00 in methanol-d 4 ) at 121 MHz.
a Chemical shifts (ppm) referenced to solvent peak (d H 7.26 in CDCl 3 ) at 500 MHz.similar ROESY correlation signals, d values (d C 74.4), and J values (d H 3.97, d, J = 8.3 Hz, H-17a, d H 3.60, s, H-17b) for key protons to compound 1 at C-17, indicating that they share the same relative configuration at C-16 and C-17.Compound 9 was named Eupfisenoid I, and the structure was assigned to be ent-atisan-16a,17-acetonide-19-hydroxy-3-one.

TABLE 4 The
UPLC-MS/MS results for sample.